**2.3. Chitosan NPs**

to induce the broadly neutralizing Abs (bnAbs), and formulating a highly efficient VADS [25]. As mentioned above, a VLP-based VADS proves a highly potent inducer for Abs and helper T cell responses and also able to elicit robust cytotoxic T cell responses necessary for preventing primary infections and erasing infected cells, thus offering researchers an alternative tool to engineer effective vaccines against HIV, which is regarded as the most challenging foe owing to its poor immunogenicity, fragile surface glycoprotein, and the ability to overpower the cell

Recently, Chapman et al*.* constructed an MVA (modified vaccinia Ankara)-mGag (an HIV-1 subtype C mosaic Gag immunogen) and a DNA-mGag vaccine, which were designed to address the tremendous diversity of HIV, and showed that mGag budded from cells infected and transfected with MVA-mGag and DNA-mGag, respectively, formed VLPs [27]. In mice, the DNA-mGag homologous prime boost vaccination elicited predominantly CD8+ T cells, and the homologous MVA-mGag vaccination induced predominantly CD4+ T cells; in contrast, a heterologous DNA-mGag prime MVA-mGag boost induced strong, more balanced Gag CD8+ and CD4+ T cell responses that were predominantly of an effector memory phenotype. Also, it was found that DNA-mGag homologous vaccination induced much higher cumulative Ag-specific IFN-γ secretion responses and generated significant higher levels of cytokine-positive CD8+ T cells than DNA-nGag (natural Gag), indicating a heterologous prime-boost regimen with DNA and MVA vaccines expressing HIV-1 subtype C mosaic Gag as an Ag is highly immunogenic and may be an effective VLP-based VADS for eliciting strong immunity to HIV. Yao's group formulated a VLP-based HIV vaccine, which was composed of HIVIIIB Gag and HIVBaL gp120/ gp41 envelope as a pseudovirion vaccine capable of presenting Ags in their native conformations and was engineered through using HEK (human embryonic kidney cell)-derived cell line expression system [28]. The researchers demonstrated that mice vaccinated by intranasal prime followed by two sub-cheek boosts with VLPs adjuvanted with liposomes entrapping TLR3 ligand dsRNA were stimulated to secrete high titers of Abs against the Ags, with predominant IgG2c over IgG and produce a significantly increased germinal center B cells and T follicular cells, suggesting that the VLP-based VADS is superior for induction of a Th1-biased immune response, while prolonging lymph node germinal centers, T follicular cells, and generating

neutralizing antibodies, and thus is rather suitable for making HIV vaccines [26].

Notably, certain types of pathogens that are once known to cause only a mild and self-healing illness and therefore never listed in dangerous items and may abruptly cause the unexpected problems associated with human and population health, hinting the existence of undisclosed infection mechanisms and pathophysiological processes or the emergence of mutations relevant to severe toxicity. For example, during the 2015–2016 South American Zika epidemic, the mosquito-borne virus which used to cause mild symptoms, such as fever, skin rash, and joint or muscle pain, was eventually identified able to cause severe damage to fetal brain through infecting pregnant women and thus finally recognized as the culprit responsible for thousands of microcephaly affected new borns, raising a great social problem and concern [29]. Unfortunately, up to now, still there are no licensed vaccines for prophylaxis of Zika, though several conventional approaches have been tried on developing such as an urgently needed products, including inactivated, recombinant live-attenuated viruses, protein subunit vaccines, RNA and DNA vaccines, as well as the VLP-based VADS [30]. Recently, using

immune system [26].

32 Immunization - Vaccine Adjuvant Delivery System and Strategies

Chitosan, a linear polysaccharide composed of randomly distributed β-(1,4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit), is usually made through hydrolysis of the chitin shells of shrimp and other crustaceans with an alkaline substance, such as sodium hydroxide [34]. Chitosan has a positive charge under neutral conditions due to protonation of basic amine groups, which contributes to the enhancement of solubility of the compound; however, the dissolution behavior of chitosan in aqueous media is also negatively influenced by the number and structural form of acetylated groups, allowing chitosan able to be used for agent delivery in several distinct forms, including solution, hydrogel, and especially nano/microparticle, which can be obtained via crosslinking, ionotropic gelation or precipitation-coacervation [35]. Interestingly, novel chitosan derivatives with customized biochemical properties are still continuously synthesized through facile conjugation of side chain moieties of functional molecules to solvent-accessible amine and hydroxyl groups, rapidly expanding chitosan in application range and dosage form [36].

In retrospect, in 1980s, researchers observed that chitosan of 70% deacetylated chitin could activate peritoneal macrophages [37] and induce production of various cytokines in mice [38], which was subsequently further explored by Illum et al*.* to demonstrate that in mouse model chitosan, via nasal immunization, could act as an efficient VADS able to remarkably enhance local as well as systemic Ab responses toward the vaccines containing filamentous hemagglutinin from *Bordetella pertussis* [39]. The researchers also proved that chitosan could significantly elevate immunogenicity of the nasal vaccine of diphtheria toxoid (DT) and enhanced vaccines in induction of high levels of Ag-specific IgG, secretory IgA, toxin-neutralizing Abs, and T cell responses, predominately of Th2 subtype [40].

be a promising VADS system for antigen delivery [49]. Interestingly, to develop a vaccine able induce robust mucosal immunity against *Chlamydia trachomatis* (Ct), which is the most common sexually transmitted infection in humans, Rose and cowokers fabricated the PLGA NPs covered with the mucoadhesive chitosan and used as a VADS for delivering recombinant Ct fusion Ag CTH522 [50]. Mice intranasally immunized with the optimized chitosan-coated PLGA NPs containing Ct Ags established potent Ag-specific systemic as well as mucosal immunity, characterized by high levels of anti-CTH522 IgG/IgA Abs in the lungs and the genital tract and high frequency of IFN-γ producing Th1 cells, suggesting that chitosan-coated PLGA NPs may be a promising mucosal VADS for delivering vaccines against sexually trans-

Polymeric Nanoparticles Engineered as a Vaccine Adjuvant-Delivery System

http://dx.doi.org/10.5772/intechopen.81084

35

Notably, in a randomized two center phase I clinical trial, an HIV vaccine consisting of HIV-I Clade C-CN54GP140 envelope glycoprotein was administered to HIV negative female volunteers through intramuscular (i.m.) immunization with glucopyranosyl lipid adjuvant (GLA), intranasal (i.n.) immunization with 0.5% chitosan, and intravaginal (i.va.) immunization with an aqueous gel vehicle [51]. The results indicated that, compared to subjects with i.n. or i.va. immunizations, recipients with three i.m. immunizations at the dose of either 20 or 100 μg CN54 gp140 secreted greater systemic and mucosal antibodies, but even in the i.m. immunized subjects, only modest neutralizing responses against closely matched tier 1 clade C virus were triggered; and the i.n. primed subjects were induced the strongest CD4+ T cell response, and, following additional i.m. boosting, were also induced an anamnestic antibody response, suggesting i.n. immunization of HIV vaccines formulated with chitosan may be an effective prime for i.m. boost.

Summarily, due to good tolerability, safety, and, particularly, mucosa-adhesive properties, chitosan and derivatives represent a promising polymer suitable for constructing mucosal VADS to provide great opportunity for developing mucosal vaccines against numerous pathogens which invade hosts through mainly mucosa. However, the available clinical results indicate clearly that to construct an effective vaccine with chitosan to handle the intractable pathogens, such as HIV, further efforts are needed to commit to optimizing formulation, seeking optimal

PLGA represents one of the most popular polymers for constructing a VADS due to its excellent safety profile, biodegradable properties, ease for processing NPs through double emulsion method, diverse modification to bear functional groups, and also the established use in several marketed products for controlled or targeted delivery of drugs [52]. It is now clear that in vivo PLGA hydrolyzes into metabolite monomers of lactic acid and glycolic acid, both of which are endogenous and easily metabolized by the body via the Krebs cycle, leaving behind little systemic toxicity, allowing wide use of PLGA as a VADS or DDS (drug delivery system). Notably, one of the appealing issues associated with the use of PLGA NPs as a VADS is attributed to the confirmation that, after cellular internalization via fluid phase pinocytosis or clathrin-mediated endocytosis, PLGA NPs may rapidly escape the endolysosomes and carry the loaded cargoes to cytoplasm, avoiding lysosomal degradation into null fragments and

immunization routes, as well as exploring combination with appropriate adjuvants.

mitted *Chlamydia trachomatis*.

**2.4. PLGA NPs**

thus enhancing vaccine delivery efficiency [53].

Chitosan, as a cationic polysaccharide bearing lots of reactive groups, possesses beneficial properties for vaccine formulation, including biocompatibility, flexibility in terms of formulation and degree of deacetylation, and efficacy when administered via mucosal route, and ability to promote immune responses [41, 42], and thus is thought a superior alternative to alum which only favors promoting humoral responses [43]. Also, chitosan is thought suitable for constituting a mucosal VADS thanks to its bio-adhesive character and intrinsic adjuvanticity, which may arise from chitosan-mediated inflammasome activation [44], or from triggering certain type of PRRs such as TLR4 on immune cells [45], in consistence with its parent molecule chitin which is confirmed able to activate immunocytes via binding to mannose receptors and TLR2 to initiate innate immune responses [46]. However, in spite of numerous research outcomes confirming its strong immunostimulatory potency, the exact mechanism underlying the intrinsic adjuvanticity of soluble chitosan remains yet elusive and needs to be fully discovered through deep exploration. More recently, it was demonstrated that after APC uptake, the intracellular chitosan induced mitochondrial damage, characterized by the generation of mitochondrial ROS and release of endogenous DNA into the cytosol, both of which culminated in the activation of the cytosol DNA sensor cGAS (cyclic-di-GMP-AMP synthase) and subsequent STING (stimulator of IFN gene) pathway, leading to translation of type I IFN and the type I IFN-dependent APC maturation to sponsor cellular immunoresponses [47].

In comparison to free chitosan, chitosan NPs that are design as a VADS capable of enhancing both humoral and cellular immune responses to delivered vaccines are proposed to function relevant to not only chitosan properties but also several aspects associated with NPs, including antigen protection, depot formation, enhanced antigen uptake, and presentation, triggering APCs via different pathways to regulate immune reaction pathways [41]. Recently, Dhakal et al. using the ionic gelation method engineered the chitosan NPs that were loaded with killed swine influenza A H1N2 virus (KIV-CNPs) and demonstrated that the nursery pigs intranasally vaccinated with KIV-CNPs produced high levels of systemic IgG and secretory IgA in nasal mucosa, bronchoalveolar lavage fluids, and lung lysates, which, more importantly, were cross-reactive against homologous (H1N2), heterologous (H1N1), and heterosubtypic (H3N2) influenza A virus strains [48]. Also, the vaccinated pigs demonstrated high frequency of Ag-specific CTLs and lymphocyte proliferation, and stimulation-recalled IFN-γ secretion, leading these pigs to experience reduced severity of macroscopic and microscopic influenza-associated pulmonary lesions after challenge with heterologous viruses, firmly confirming that the NPs composed of chitosan may function as an effective mucosal VADS favoring noninvasive immunization. In another report, Lebre et al. prepared chitosan-aluminum nanoparticles (CH-Al NPs) with a size of 280 nm and a positive surface charge and proved that CH-Al NPs loaded with hepatitis B surface antigen (HBsAg) were more stable in physiological environment and more efficient in inducing cellular immunity than common chitosan NPs, suggesting the two combination immunostimulants chitosan and aluminum salts may be a promising VADS system for antigen delivery [49]. Interestingly, to develop a vaccine able induce robust mucosal immunity against *Chlamydia trachomatis* (Ct), which is the most common sexually transmitted infection in humans, Rose and cowokers fabricated the PLGA NPs covered with the mucoadhesive chitosan and used as a VADS for delivering recombinant Ct fusion Ag CTH522 [50]. Mice intranasally immunized with the optimized chitosan-coated PLGA NPs containing Ct Ags established potent Ag-specific systemic as well as mucosal immunity, characterized by high levels of anti-CTH522 IgG/IgA Abs in the lungs and the genital tract and high frequency of IFN-γ producing Th1 cells, suggesting that chitosan-coated PLGA NPs may be a promising mucosal VADS for delivering vaccines against sexually transmitted *Chlamydia trachomatis*.

Notably, in a randomized two center phase I clinical trial, an HIV vaccine consisting of HIV-I Clade C-CN54GP140 envelope glycoprotein was administered to HIV negative female volunteers through intramuscular (i.m.) immunization with glucopyranosyl lipid adjuvant (GLA), intranasal (i.n.) immunization with 0.5% chitosan, and intravaginal (i.va.) immunization with an aqueous gel vehicle [51]. The results indicated that, compared to subjects with i.n. or i.va. immunizations, recipients with three i.m. immunizations at the dose of either 20 or 100 μg CN54 gp140 secreted greater systemic and mucosal antibodies, but even in the i.m. immunized subjects, only modest neutralizing responses against closely matched tier 1 clade C virus were triggered; and the i.n. primed subjects were induced the strongest CD4+ T cell response, and, following additional i.m. boosting, were also induced an anamnestic antibody response, suggesting i.n. immunization of HIV vaccines formulated with chitosan may be an effective prime for i.m. boost.

Summarily, due to good tolerability, safety, and, particularly, mucosa-adhesive properties, chitosan and derivatives represent a promising polymer suitable for constructing mucosal VADS to provide great opportunity for developing mucosal vaccines against numerous pathogens which invade hosts through mainly mucosa. However, the available clinical results indicate clearly that to construct an effective vaccine with chitosan to handle the intractable pathogens, such as HIV, further efforts are needed to commit to optimizing formulation, seeking optimal immunization routes, as well as exploring combination with appropriate adjuvants.

#### **2.4. PLGA NPs**

local as well as systemic Ab responses toward the vaccines containing filamentous hemagglutinin from *Bordetella pertussis* [39]. The researchers also proved that chitosan could significantly elevate immunogenicity of the nasal vaccine of diphtheria toxoid (DT) and enhanced vaccines in induction of high levels of Ag-specific IgG, secretory IgA, toxin-neutralizing Abs,

Chitosan, as a cationic polysaccharide bearing lots of reactive groups, possesses beneficial properties for vaccine formulation, including biocompatibility, flexibility in terms of formulation and degree of deacetylation, and efficacy when administered via mucosal route, and ability to promote immune responses [41, 42], and thus is thought a superior alternative to alum which only favors promoting humoral responses [43]. Also, chitosan is thought suitable for constituting a mucosal VADS thanks to its bio-adhesive character and intrinsic adjuvanticity, which may arise from chitosan-mediated inflammasome activation [44], or from triggering certain type of PRRs such as TLR4 on immune cells [45], in consistence with its parent molecule chitin which is confirmed able to activate immunocytes via binding to mannose receptors and TLR2 to initiate innate immune responses [46]. However, in spite of numerous research outcomes confirming its strong immunostimulatory potency, the exact mechanism underlying the intrinsic adjuvanticity of soluble chitosan remains yet elusive and needs to be fully discovered through deep exploration. More recently, it was demonstrated that after APC uptake, the intracellular chitosan induced mitochondrial damage, characterized by the generation of mitochondrial ROS and release of endogenous DNA into the cytosol, both of which culminated in the activation of the cytosol DNA sensor cGAS (cyclic-di-GMP-AMP synthase) and subsequent STING (stimulator of IFN gene) pathway, leading to translation of type I IFN and the type I IFN-dependent APC maturation to sponsor cellular immunoresponses [47].

In comparison to free chitosan, chitosan NPs that are design as a VADS capable of enhancing both humoral and cellular immune responses to delivered vaccines are proposed to function relevant to not only chitosan properties but also several aspects associated with NPs, including antigen protection, depot formation, enhanced antigen uptake, and presentation, triggering APCs via different pathways to regulate immune reaction pathways [41]. Recently, Dhakal et al. using the ionic gelation method engineered the chitosan NPs that were loaded with killed swine influenza A H1N2 virus (KIV-CNPs) and demonstrated that the nursery pigs intranasally vaccinated with KIV-CNPs produced high levels of systemic IgG and secretory IgA in nasal mucosa, bronchoalveolar lavage fluids, and lung lysates, which, more importantly, were cross-reactive against homologous (H1N2), heterologous (H1N1), and heterosubtypic (H3N2) influenza A virus strains [48]. Also, the vaccinated pigs demonstrated high frequency of Ag-specific CTLs and lymphocyte proliferation, and stimulation-recalled IFN-γ secretion, leading these pigs to experience reduced severity of macroscopic and microscopic influenza-associated pulmonary lesions after challenge with heterologous viruses, firmly confirming that the NPs composed of chitosan may function as an effective mucosal VADS favoring noninvasive immunization. In another report, Lebre et al. prepared chitosan-aluminum nanoparticles (CH-Al NPs) with a size of 280 nm and a positive surface charge and proved that CH-Al NPs loaded with hepatitis B surface antigen (HBsAg) were more stable in physiological environment and more efficient in inducing cellular immunity than common chitosan NPs, suggesting the two combination immunostimulants chitosan and aluminum salts may

and T cell responses, predominately of Th2 subtype [40].

34 Immunization - Vaccine Adjuvant Delivery System and Strategies

PLGA represents one of the most popular polymers for constructing a VADS due to its excellent safety profile, biodegradable properties, ease for processing NPs through double emulsion method, diverse modification to bear functional groups, and also the established use in several marketed products for controlled or targeted delivery of drugs [52]. It is now clear that in vivo PLGA hydrolyzes into metabolite monomers of lactic acid and glycolic acid, both of which are endogenous and easily metabolized by the body via the Krebs cycle, leaving behind little systemic toxicity, allowing wide use of PLGA as a VADS or DDS (drug delivery system). Notably, one of the appealing issues associated with the use of PLGA NPs as a VADS is attributed to the confirmation that, after cellular internalization via fluid phase pinocytosis or clathrin-mediated endocytosis, PLGA NPs may rapidly escape the endolysosomes and carry the loaded cargoes to cytoplasm, avoiding lysosomal degradation into null fragments and thus enhancing vaccine delivery efficiency [53].

To develop an effective VADS, Noormehr et al*.* fabricated 500-nm-sized PLGA NPs which were covalently conjugated with recombinant Ags Leishmanial CPA (cysteine peptidase A) and CPB, and proved that mice intra-peritoneally immunized with the inhomogeneous Ag-NPs secreted high levels of NO (nitric oxide) by peritoneal MPs and high levels of IFN-γ by splenocytes, which significantly lowered *Leishmania major* burden, suggesting the Ag-conjugated PLGA NPs can be used as a VADS able to deliver vaccines to protect against the tough pathogen of parasites [54]. To investigate the function of multiple adjuvant-combined VADS, Ebrahimian and colleagues formulated the TLR 7/8a resiquimod- or TLR4a MPLA-loaded PLGA NPs which were physically covered with polyethylenimine (PEI) forming PLGA/PEI NPs and then mixed with CpG ODN (cytosine-phosphorothioate-guanine oligodeoxynucleotide) to engender a complexed entity of resiquimod- or MPLA-PLGA NPs/PEI-CpG ODN [55]. Given to BALB/c mice, the multiple adjuvant-constituted PLGA NPs loaded with Ags induced robust and efficient immune responses, as confirmed by evaluation of vivo cytokine (IFN-γ, IL-4, and IL-1β) secretion and antibody (IgG1 and IgG2a) production, demonstrating using a combination of adjuvants in a context-dependent manner may a feasible strategy for engineering a potent PLGA-based VADS. To make subunit vaccines suitable for immunization via skin, which is an attractive but also very challenging immunization site due to the presence of affluent APCs while difficulty of administration, recently, Bouwstra's group fabricated the hyaluronan (HA)-based dissolving microneedles (MNs) entrapped with PLGA NPs which co-encapsulated ovalbumin (OVA) as an Ag and poly(I:C) as an adjuvant for intradermal immunization [56]. Further investigation indicated that the immunogenicity of the PLGA NPs after administration of dissolving MNs was compared with that of hollow MN-delivered PLGA NPs in mice, while immunization with free Ag in dissolving MNs resulted in equally strong immune responses compared to delivery by hollow MNs. However, humoral and cellular immune responses evoked by PLGA NP-loaded dissolving MNs were inferior to those elicited by NPs delivered through a hollow MN, suggesting several critical parameters should be fully evaluated in engineering the PLGA NP-loaded dissolving MNs as an intradermal VADS to avoid unnecessary efforts on the complexed formulations.

multiple injections of liquid vaccine, suggesting the VADS constructed with the bPEI-PLGA

Polymeric Nanoparticles Engineered as a Vaccine Adjuvant-Delivery System

http://dx.doi.org/10.5772/intechopen.81084

Conclusively, PLGA is a biodegradable, safe, and clinically used polymer, which, using the double-emulsion method, can be conveniently engineered into NPs to constitute a VADS with appropriate features and abilities to render vaccine lysosome escape, thus enhancing vaccina-

Hyaluronic acid (HA) as a polysaccharide consists of alternating units of D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (Glc-NAc), connected to each other with β-1,3- and β-1,4 glycosidic bonds, having nearly perfect chemical repeats except for occasional deacetylated glucosamine residues to form a very hydrophilic linear high molecular weight (HMW) biopolymer [59]. HA ranges in size from 5 kDa to 10 million Da (corresponding to 25,000 disaccharide units), with the most common forms of 1–8 million Da in humans and can absorb water to expand its solid volume by up to 1000 times forming a very viscous and elastic gel [60]. HA GlcA carboxyl group is dissociated at physiological pH values to engender a negatively charged

polymer which is readily combining with the most prevalent extracellular cation of Na<sup>+</sup>

sodium hyaluronate, suggesting that the molecule is not ionized [59]. In fact, while native HA with a high molecular weight (HMW) acts mainly as a constructive stuff and a control on tissue hydrodynamics, low molecular weight (LMW) HA usually participates in cell signaling through interaction with certain types of cell surface receptors, such as CD44 as the primary one, but also TLR2 and TLR4, thus contributing to several physiological and biological activities [61, 62]. As an abundant endogenous polymer, HA has been widely exploited to construct the functional carriers for delivering various bioactives with expectation of improving human health, given many of its desirable merits that can be employed for optimizing delivery effects [63]. Most attractively, HMW HA possesses numerous physicochemical and physiological features, such as biocompatibility, biodegradation, mucoadhesive property, bearing negative charges in a neutral condition, possessing active groups allowing various functional modifications that are all beneficial characters for engineering carriers to deliver agents [64]. Another interesting aspect lies in that LMW HA binds to several receptors, especially CD44, which is ubiquitously expressed on various cells, and especially overexpressed on many types of cancer cells, presenting bases for developing a tumor-targeting drug delivery system (DDS) with attractive advantages, such as the ease of associating drugs with the polysaccharide or its carrier thus solving any solubility problems, improving a drug's blood plasma half-life thus playing a similar role to PEG, and high tumor-targeting efficiency, and as such is currently the main trend in the HA-based delivery research [65]. More recently, LMW HA is focused on its ability to activate CD44 or TLRs on immune cells involving regulation of certain signaling pathways associated with APC maturation, cytokine production and innate immune responses for immunization, and even, in a CD44- and TLR4-independent manner, to enhance CCR7 expression on DCs promoting DC recruitment to tumor regional lymph nodes and restraining DC migration toward tumor tissue [62, 66–68], thus providing not only a comprehensive option for engineering functional nanoparticles fitting a VADS, but also a promising candidate for improving DC maturation in the context of DC-based vaccine development.

to form

37

microparticles has big potential to elevate vaccine coverage in the developing world.

tion efficiency.

**2.5. Hyaluronic acid (HA)-modified liposomes**

At present, still a large fraction of vaccines require a multiple dosing schedule with a 1- to 2-month gap between administrations to guarantee establishing the Ag-specific immunity strong enough to protect recipients, as such, however, engendering a big challenge to worldwide vaccination, especially, in the developing countries, where healthcare workers are not only in shortage but also confronting difficulty in reaching the subjects multiple times to administer booster shots [57]. Conceptually, this challenge may be conquered using a VADS that are constructed with a functional carrier which release vaccine ingredients in pulses with an appropriate time gap between vaccinations, thus simplifying the vaccination schedule to consist of only once injection to exclude additional visits by a healthcare worker. For this, Tzeng et al*.* engineered a controlled release VADS consisting of bPEI (branched PEI)-modified PLGA microparticles which contained in inner core Ags of IPV (inactivated polio vaccine with three antigens) and an Ag stabilizer poly(L-lysine) [58]. Further investigation indicated that the bPEI-PLGA microparticles stabilized IPV in its active conformation inside the particles for months but in an aqueous medium released two bursts of IPV with an interval of just 1 month, vividly mimicking a typical twice vaccination schedule. Moreover, one injection of the controlledrelease formulations elicited a similar or better Ag neutralizing response in rats compared to multiple injections of liquid vaccine, suggesting the VADS constructed with the bPEI-PLGA microparticles has big potential to elevate vaccine coverage in the developing world.

Conclusively, PLGA is a biodegradable, safe, and clinically used polymer, which, using the double-emulsion method, can be conveniently engineered into NPs to constitute a VADS with appropriate features and abilities to render vaccine lysosome escape, thus enhancing vaccination efficiency.
